The present invention relates to a method for synthesizing SERS (Surface-Enhanced Raman Scattering) encoded nanoparticles. These particles are useful in different fields such as high-throughput multiplex screening[1] in microarray technology,[2] diagnosis[3] and bioimaging.[4]
Encoded nanoparticles are between the most powerful alternatives for high-throughput multiplex screening[1] in microarray technology,[2] diagnosis[3] and bioimaging.[4] These materials are simple and cost-effective platforms which allow for fast, sensitive and reliable analysis.[1b, 5] During the last decade, several encoded particles were prepared[6] using codification strategies based on changes in particle shape,[7] composition,[8] physical marks[6c] or spectroscopic properties (e.g. luminescence or vibrational fingerprints).[4, 9] Among all of them, those based on Surface-Enhanced Raman Scattering (SERS) are gaining importance[10] due to: i) the virtually unlimited multiplexing capability associated with the unique vibrational fingerprint of the different codes, ii) short detection times (milliseconds) thanks to the intrinsic sensitivity of the SERS phenomena;[11] iii) small size, allowing for bioimaging;[12] and, iv) photostability and low toxicity (as compared with those of dyes or quantum dots).[13]
In essence, a SERS-encoded nanoparticle (also indicated as SERS-tag) comprises a plasmonic nucleus, responsible for the generation of the electric field necessary for the Raman amplification; a Raman probe (i.e. code), responsible of the unique vibrational fingerprint of the encoded particle; and, a coating layer. This external coating is of key importance as: i) prevents the code from leaching out into the medium thus avoiding toxic effects or vibrational cross-contamination with the codes of other particles; ii) protects the plasmonic particle from contaminations of the medium that may give rise to vibrational noise hindering the particle readout; iii) increases the colloidal stability of the particle; iv) provides a convenient surface for further chemical functionalization; and, v) protects the plasmonic core for interacting with other plasmonic particles avoiding plasmon coupling and so the uncontrolled generation of hotspots. Although, polymers, have been reported as particles coatings[12b, 14] the unique properties of silica (i.e. known surface chemistry, biocompatibility, optical transparency and colloidal stability) make this material the most efficient protective layer for nanoparticles by far.[15] US2006054506A1 discloses a method for synthesizing encapsulated SERS encoded nanoparticles in which a selected SERS encoding molecule is added to an aqueous suspension of metal nanoparticles, and after that, the SERS encoding nanoparticles are encapsulated in a silica matrix. However, this procedure is restricted only to a limited number of encoding molecules, those containing already pyridyl or silane groups, because these groups can act as silica precursors.
Silica coating of nanoparticles requires the colloidal stabilization of the particles in ethanolic solution prior to the hydrolysis/condensation of tetraethyl orthosilicate (TEOS). Though a range of polymers have been described for this task,[15a, 16] the most common remains polyvinylpyrrolidone (PVP).[15a, 16] On the other hand, surfactants such as cetyltrimethyl ammonium bromide (CTAB) also are used commonly for this reaction.[15a] Notwithstanding, the fact that the most important factor for the generation of active SERS-encoded particles relies in the intimate contact between the Raman code and the plasmonic particle, introduces further complexity to the coating process associated with the surface chemistry properties. Both PVP and CTAB form solid layers of coating on the surface of the particles limiting or even avoiding the interaction of the encoding agent with the metallic surface when added to the solution.[17] Therefore, to increase the code adsorption efficiency on the plasmonic structure, and thus the SERS signal, PVP and CTAB species need to be removed from the metallic surfaces. On the other hand, this usually results in a drastic reduction of the colloidal stability, which is further aggravated by the non-polar nature of most of the codes, leading to uncontrolled particle agglomeration[11, 18] or even to irreversible precipitation. Aggregation of labelled-nanoparticles into clusters of different size and geometry does generate very active SERS structures but with highly inhomogeneous SERS response. Moreover, these fabrication methods normally work for a very limited number of encoding molecules as, in many cases, precipitation of the whole colloids occurs upon addition of the code. In fact, this explains why in most of the literature, examples of SERS encoded particles include a small number of codes, usually just three or four.
As an alternative to the conventional polymers or surfactants, thiolated poly(ethylene glycol) (PEG) had been successfully employed for the controlled silica coating of single metallic nanoparticles. The high polarity and porosity of this polymer efficiently stabilize particles in alcohol and water while allow for the diffusion of the code to the metallic surface.[19] No matter, as commonly in polymers, PEG size distribution normally suffers from large fluctuations from batch-to-batch, even for the same commercial brand. As a result, the synthetic protocol to encode particles using this method needs to be tuned every time a new PEG is purchased. Additionally, the high price of the thiolated-PEG hinders its use to the large-scale preparation of encoded particles as required for real life applications.